Method of processing lignocellulosic feedstock for enhanced xylose and ethanol production

ABSTRACT

The present invention provides a method of producing xylose from lignocellulosic feedstock. The method comprises disrupting lignocellulosic feedstock; leaching the lignocellulosic feedstock by contacting the feedstock with at least one aqueous solution for a period greater than about 2 minutes to produce a leached feedstock and a leachate; removing the leachate from the leached feedstock; acidifying the leached feedstock to a pH between about 0.5 and about 3 to produce an acidified feedstock, and; reacting the acidified feedstock under conditions which disrupt fiber structure and hydrolyze a portion of hemicellulose and cellulose of the acidified feedstock, to produce a composition comprising xylose and a pretreated feedstock. The xylose may be purified from the pretreated feedstock or it may be converted to ethanol with the pretreated feedstock.

This application is the U.S. national phase application of PCTInternational application No. PCT/CA02/00244, which claims the benefitof U.S. provisional application No. 60/272,353, filed on Feb. 28, 2001.

The present invention relates to pretreatment processes for theconversion of lignocellulosic feedstocks into sugars. More specifically,the present invention relates to pretreatment processes for theconversion of lignocellulosic feedstocks into sugars and the subsequentconversion of sugars to ethanol.

BACKGROUND OF THE INVENTION

The possibility of producing ethanol from cellulose-containinglignocellulosic feedstocks such as wood, cultivated crops like switchgrass and waste agricultural fibers such as wheat straw and oat hullshas received much attention due to the availability of large amounts offeedstocks, the desirability to avoid burning or landfilling cellulosicwaste materials, and the cleanliness of ethanol as a fuel compared togasoline.

The efficient conversion of cellulose from lignocellulosic material intoglucose and the subsequent fermentation of glucose to ethanol representsa major challenge. Cellulose, which is the primary constituent oflignocellulosic fibers, consists of a crystalline structure that is veryresistant to breakdown, as is hemicellulose, the second most prevalentcomponent. The conversion of lignocellulosic fibers to ethanolrequires: 1) liberating cellulose and hemicellulose from lignin orincreasing the accessibility of cellulose and hemicellulose within thelignocellulosic feedstock to cellulase enzymes, 2) depolymerizinghemicellulose and cellulose carbohydrate polymers to free sugars and, 3)fermenting the mixed hexose and pentose sugars to ethanol.

Methods used to convert cellulose to glucose typically include acidhydrolysis (reviewed by Grethlein; Chemical Breakdown Of CellulosicMaterials, J.APPL.CHEM. BIOTECHNOL. 28:296-308 (1978)). Acid hydrolysisinvolves the use of either concentrated or dilute acids. Theconcentrated acid process uses 72% by weight sulfuric acid or 42% byweight hydrochloric acid at room temperature to dissolve the cellulose,followed by dilution to 1% acid and heating to 100° C. to 120° C. for upto three hours to convert cellulose oligomers to glucose monomers. Thisprocess produces a high yield of glucose, but the recovery of the acid,and the specialized construction materials required for the apparatus tocarry out this process are serious disadvantages. Similar problems areencountered when concentrated organic solvents are used for celluloseconversion.

U.S. Pat. No. 5,536,325 describes a two-step process for the acidhydrolysis of lignocellulosic material to glucose. The first (mild) stepdepolymerizes the hemicellulose to xylose and other sugars. The secondstep depolymerizes the cellulose to glucose. Even though the processuses low levels of acid, the amount of acid required for the hydrolysisof the feedstock is substantial, and the resulting yield of glucose fromcellulose is poor.

Other methods for converting lignocellulosic material to ethanol use amultistep procedure in which the lignocellullosic material is firstpretreated at high temperature and pressure and often in the presence ofvarious chemicals. This pretreatment process is thought to increase theaccessibility of cellulose within the lignocellulosic fibers forsubsequent conversion steps. As a large portion of the cellulose withinuntreated lignocellulosic material is unaccessible for subsequentenzymatic conversion steps, the efficiency of this pretreatment phasecan profoundly influence the overall efficiency and commercialapplication of the entire conversion process.

Among the more successful pretreatment processes for the conversion oflignocellulosic feedstock into glucose, are dilute acid prehydrolysisprocesses. One effective dilute acid hydrolysis pretreatment is steamexplosion as disclosed in U.S. Pat. No. 4,461,648 (Foody process, whichis herein incorporated by reference). In the Foody process, biomass isloaded into a vessel known as a steam gun. Up to 1% acid is optionallyadded to the biomass in the steam gun or in a presoak. The steam gun isthen filled very quickly with steam and held at high pressure for a setlength of time, known as the cooking time. Once the cooking timeelapses, the vessel is depressurized rapidly to expel the pretreatedbiomass. As a result of the rapid depressurization, the Foody processhas been termed “steam explosion”. Specific parameters for steamexplosion pretreatments are set out in U.S. Pat. No. 4,461,648; andFoody, et al, Final Report, Optimization of Steam ExplosionPretreatment, U.S. DEPARTMENT OF ENERGY REPORT ET230501 (Apr. 1980),which are herein incorporated by reference.

U.S. Pat. No. 4,237,226 describes the dilute-acid pretreatment of oak,newsprint, poplar, and corn stover by a continuous plug-flow reactor, adevice that is similar to an extruder. Rotating screws convey afeedstock slurry through a small orifice, where mechanical and chemicalaction break down the fibers to increase the accessibility to cellulose.

One shortcoming of dilute acid prehydrolysis is the high acidrequirement. For a clean feedstock, such as washed hardwood, thesulfuric acid demand is 0.5% to 1% of the dry weight of the feedstock.For agricultural fibers, which can contain high levels of silica, salts,and alkali potassium compounds from the soil, the acid demand is about10-fold higher, reaching 5% to 7% by weight of feedstock. This addssignificant additional cost to the process. A second drawback of usinglarge amounts of acids in a prehydrolysis process is that an acidifiedfeedstock must be neutralized to a pH between about 4.5 and about 5prior to enzymatic hydrolysis with cellulase enzyme. The amount ofcaustic soda used to neutralize acidified feedstock is proportional tothe amount of acid used to acidify the feedstock. Thus, high acid usageresults in high caustic soda usage, which further increases the cost ofprocessing lignocellulosic feedstock to ethanol.

Another drawback of steam explosion and other dilute acid pretreatmentprocesses is that the while the treatment conditions significantlyincrease accessibility to cellulose, these same conditions result in thedestruction and loss of xylose. Xylose is not as stable as the othersugars and has a tendency to break down in acid pretreatment conditions.The breakdown of xylose decreases the overall sugar yield that can beobtained from lignocellulosic feedstocks and this in turn decreasesethanol yield.

U.S. Pat. No. 5,198,074 and U.S. Pat. No. 4,857,145 (which are hereinincorporated by reference) disclose washing chiped feedstock with waterprior to removing a soluble fraction, used for the production ofethanol, and a fibre fraction for use in pulp and paper production.There is no disclosure of the use of the pretreated fraction for theproduction of xylose or ethanol.

U.S. Pat. No. 5,846,787 discloses processes for pretreating cellulosicmaterials prior to enzymatic conversion with cellulases. The processinvolves heating the cellulosic materials in water at a temperature ator above their glass transition temperature while maintaining the pH ofthe reaction medium in a range that avoids autohydrolysis of thecellulosic materials. The method is performed in place of a dilute acidor steam explosion pretreatment process. The water used in thepretreatment process of U.S. Pat. No. 5,846,787 is heated under pressureto a temperature in excess of 100° C., and thus requires a significantamount of energy to heat the water to such temperatures and is expensiveand inefficient.

U.S. Pat. No. 4,326,892 discloses a method of improving the recovery ofsugar from sugar beets. Sugar beets contain primarily sucrose, withlittle cellulose. The method comprises washing sugar beets to removeimpurities therefrom, removing the outer layer of the of the washedsugar beets, slicing the sugar beets and extracting the slice sugarbeets with an aqueous solution. After this washing, the relative amountof cellulose in the resulting beet pulp is increased. There is nosuggestion that the pulp obtained by this process may be used for theproduction of xylose or ethanol as described herein.

U.S. Pat. No. 6,251,643 discloses a method for processing biomass usinga screw press. In this method, following separation of a liquid phasefrom the solid phase, the solid phase is heated under pressure, to atemperature of 100-170° C., to produce a vapour treated phase of solidswhich is then further processed. The use of high temperatures to producethe vapour treated phase results in the denaturing of any proteincomponent within the solid phase. Furthermore, the process described inthis document results in a relatively low yield of ethanol from biomass(180-250 liters ethanol per metric ton dry material).

The publication entitled Wet Milling of Grain for Alcohol Production byLyons et al., in Chapter 1 of The Alcohol Textbook 1995 (NottinghamUniversity Press) discloses wet milling of corn in alcohol production.The corn kernel comprises approximately 70% starch and contains littlecellulose. After starch removal, the resulting corn fiber is high incellulose. The reference teaches soaking clean corn in tanks for about20 to 40 hours with steep water/acid containing about 1600 ppm SO₂, at atemperature of about 52° C. This steeping is the first step in removingstarch from corn. However, the reference does not teach the use of thecorn fiber for the production of xylose or ethanol as described herein.

The publication entitled “Separation Processes” by C. Judson King (1980,McGraw-Hill Book Company) discloses processing of sugar cane. Sugar caneis primarily sugar, with little cellulose. The reference teaches washingsugar cane with jets of water to remove field debris, followed bychopping the sugar cane into short sections, passing the sectionsthrough high pressure rollers and adding water to remove the availablesugar. The sugar solution is processed and refined into raw sugar, blackmolasses and other products. After sugar removal, the remaining canepulp (bagasse) is high in cellulose content. There is no suggestion thatthe remaining bagasse maybe used for xylose or ethanol production asoutline herein.

Jenkins et al., (Measurements of the Fouling and SlaggingCharacteristics of Banagrass (Pennisetum purpureum) Following AqueousExtraction of Inorganic Constituents. In: Making a Business from Biomassin Energy, Environment, Chemicals, Fibers and Materials. Proceedings ofthe 3^(rd) Biomass Conference of the Americas, Montreal, Quebec, Canada,August 24-29. Pergamon (Elsevier Science)) discloses the washing ofbiomass fuel by aqueous extraction to control slagging and fouling incombustion systems burning banagrass. There is no suggestion that thewashed lignocellulosic biomass maybe used for xylose or ethanolproduction as described herein.

Methods that improve xylose yield during acid hydrolysis improve thecost efficiency of converting lignocellulosic feedstock to sugars.Furthermore, methods that reduce the amount of acid required for diluteacid pretreatments increase the cost efficiency of ethanol production.

There is a need in the art to reduce the amount of acid which must beused in a pretreatment process. Further, there is a need in the art toincrease xylose yield from lignocellulosic feedstocks subjected topretreatment processes.

It is an object of the present invention to overcome disadvantages ofthe prior art.

The above object is met by a combination of the features of the mainclaims. The sub claims disclose further advantageous embodiments of theinvention.

SUMMARY OF THE INVENTION

The invention relates to pretreatment processes for the conversion oflignocellulosic feedstocks into sugars. More specifically, the presentinvention relates to pretreatment processes for the conversion oflignocellulosic feedstocks into sugars and the subsequent conversion ofsugars to ethanol.

According to an embodiment of the present invention there is provided amethod of producing xylose from lignocellulosic feedstock comprisinggreater than about 20% (w/w) cellulose. The method comprises

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock; and    -   c) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock.

The present invention relates to the above method, wherein following thestep of removing (step b)), is a step of acidifying the leachedfeedstock to a pH between about 0.5 and about 3 to produce an acidifiedfeedstock.

The lignocellulosic feedstock may be selected from one or more of thefollowing C4 grasses: switch grass, cord grass, rye grass, miscanthus,and a combination thereof, or sugar cane bagasse, soybean stover, cornstover, rice straw, rice hulls, barley straw, corn cobs, wheat straw,oat hulls, corn fiber, wood fiber, or a combination thereof. Preferablythe lignocellulosic feedstock comprises an AX/NSP ratio of about 0.35 toabout 0.45, such as wheat straw, wheat chaff, switch grass, corn stover,corn cobs, oat hulls, or a combination thereof. The lignocellulosicfeedstock may also comprise newsprint, cardboard, sawdust andcombinations thereof. More preferably the lignocellulosic feedstockcomprises oat hulls, wheat straw, switch grass, or a combinationthereof.

Also according to the method of the present invention as defined above,the composition comprising xylose may comprise xylose polymer, xylosemonomer or a combination thereof. The composition may comprise othercomponents such as salts, sugars and the like.

Further, according to the method of the present invention as definedabove, the lignocellulosic feedstock preferably comprises mechanicallydisrupted feedstock. Preferably, the lignocellulosic feedstock ismechanically disrupted to pass through 20 mesh, more preferably 40 mesh.Mechanical disruption maybe performed by shredding, milling, chopping,chipping, grinding or a combination thereof. Preferably, mechanicaldisruption is performed by Hammer milling, Wiley milling, Szego millingor a combination thereof.

Also according to the method of the present invention as defined above,leaching is performed for a period between about 2 minutes and about 2hours, and at a temperature between about 5° C. and about 95° C.Preferably the leaching is performed at a temperature between about 20°C. and about 80° C., and more preferably between about 60° C. and about80° C. Furthermore, the aqueous solution employed in leachinglignocellulosic feedstock comprises about 0.25 to about 10 times themaximum water holding capacity per kilogram of dry lignocellulosicfeedstock. Leaching may also comprise a plurality of leaching stages. Inembodiments of the method of the present invention wherein leachingcomprises multiple Teachings, preferably leaching comprises about 2 toabout 4 leaching stages. Further, the leaching stages may be performedin a countercurrent fashion.

Also according to the method of the present invention as defined above,the aqueous solution may comprise plant water, process water, freshwater or a combination thereof. Preferably the aqueous solutioncomprises a solution of pH about 3 to about 9, and contains less thanabout 10 g/l of dissolved impurities. The pH may be adjusted using NaOH,H₂SO₄ or a combination thereof.

Further, according to the method of the present invention as definedabove, leaching preferably removes between about 50% to about 100%,preferably 70% to 100% of the total leachable buffering agents from thelignocellulosic feedstock. The leachate may be sampled during leaching,following leaching or both, and the leaching parameters, such asleaching time, or the number of leaching stages in a multistage leachingmay be adjusted accordingly.

According to the present invention there is provided a compositionproduced by the method of the present invention as defined above,wherein the composition comprises xylose and a pretreated feedstock.Preferably, the lignocellulosic feedstock yields xylose in the range ofabout 150 mg to about 260 mg of xylose per gram of feedstock. Thepretreated feedstock has increased accessibility to being digestedduring a treatment with cellulase enzyme. Further, the compositionproduced by the method of the present invention, the pretreatedfeedstock or both may be treated with cellulase enzyme to convertcellulose to glucose, and this treatment may be followed by fermentingglucose to products such as, but not limited to ethanol, or lactic acid.Glucose may also be chemically hydrogenated to sorbitol or converted toother products such as acetic acid, citric acid, ascorbic acid,propanediol, butanediol, acetone, butanol, or a combination thereof.

Also according to the method of present invention as defined above, thelignocellulosic feedstock preferably comprises cellulose in an amountgreater than about 20%, more preferably greater than about 30%, stillmore preferably greater than about 40% (w/w). The lignocellulosicfeedstock may also comprise lignin in an amount greater than about 5%,more preferably in an amount greater than about 10% (w/w). Thelignocellulosic feedstock may also comprise a combined amount ofsucrose, fructose and starch in an amount less than about 20%,preferably less than about 10% (w/w). The weight percentages disclosedabove are relative to the mass of the lignocellulosic feedstock as itexists in its natural state, prior to any processing.

The present invention also provides a method of producing xylose fromlignocellulosic feedstock comprising,

-   -   a) leaching a mechanically disrupted lignocellulosic feedstock,        the leaching comprising contacting the feedstock with at least        one aqueous solution for a period of about 2 minutes to about 2        hours at a temperature of about 20° C. to about 80° C. using a        volume of aqueous solution which is between about 0.25 and about        10 times the maximum water holding capacity of the disrupted        lignocellulosic feedstock, to produce a leached feedstock and a        leachate;    -   b) removing the leachate from the leached feedstock;    -   c) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   d) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock.

Also according to the present invention, there is provided a method ofproducing ethanol from a lignocellulosic feedstock comprising greaterthan about 20% (w/w) cellulose, the method comprising,

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock;    -   c) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   d) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock;    -   e) treating the composition comprising xylose and pretreated        feedstock with cellulase under conditions which hydrolyse        cellulose in the pretreated feedstock to glucose, producing a        sugar solution comprising xylose and glucose, and;    -   f) fermenting the sugar solution to ethanol.

Also according to the present invention, there is provided a method ofproducing ethanol from a lignocellulosic feedstock comprising greaterthan about 20% (w/w) cellulose, the method comprising,

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock;    -   c) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   d) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock, and;    -   e) treating the composition with a microrganism under conditions        which permit fermentation of the xylose in the composition to        ethanol.

Also according to the present invention, there is provided a method ofproducing ethanol from a lignocellulosic feedstock comprising greaterthan about 20% (w/w) cellulose, the method comprising,

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock;    -   c) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   d) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock;    -   e) separating the pretreated feedstock from the composition;    -   f) treating the pretreated feedstock with cellulase under        conditions which hydrolyse cellulose in said pretreated        feedstock to glucose, producing a sugar solution comprising        glucose, and;    -   g) fermenting the sugar solution to ethanol.

Thus, it is contemplated that xylose produced in the pretreatment oflignocellulosic feedstock may be fermented in combination with glucose,or separate from glucose produced by cellulose hydrolysis of thepretreated feedstock.

The present invention also provides a method for preparing alignocellulosic feedstock comprising:

-   -   a) preconditioning the lignocellulosic feedstock to produce a        preconditioned feedstock;    -   b) incubating the preconditioned feedstock within a leaching        bath to produce a leached feedstock; and    -   c) crushing the leached feedstock to produce a pressate and a        solid feedstock.

In the method as described above, the step of preconditioning (step a))may comprise heating the lignocellulosic feedstock by steam to about 80°C. for about 1 minute, and in the step of incubating (step b)), debrismay be removed from the leaching bath. Furthermore, in the step ofincubating (step b)), the preconditioned feedstock may be incubated fromabout 5 to about 30 minutes, and wherein in the step of crushing (stepc)), the pressate comprises soluble protein which is further processedfor animal feed.

The present invention also pertains to a lignocellulosic feedstockprocessing system comprising, a feedstock handling device; apreconditioner capable of receiving the feedstock from the handlingdevice, the preconditioner in communication with a steam source, thesteam source for heating the feedstock, a first transfer device, forconveying the feedstock within, and from, the preconditioner to aleaching bath, a second transfer device for mixing the feedstock withinthe leaching bath and conveying the feedstock from the leaching bath toa press, a third transfer device for conveying pressed feedstock fromthe press to a second leach stage, and multi-press, to produce processedlignocellulosic feedstock.

Using the methods as described herein for the processing of biomass,high yields of ethanol can be obtained from about 300-340 liter ethanolper metric ton biomass. Another advantage of the method as describedherein, over prior art methods for the preparation of lignocellulosicfeedstock for xylose, ethanol, or both xylose and ethanol production, isthat protein is recovered from the feedstock prior to any steam and acidtreatments. These steam and acid treatments denature protein within thefeedstock, and hydrolyse labile amino acids, for example but not limitedto lysine, that are important for high quality animal feeds. As theprotein is recovered from the feedstock processed according to thepresent invention prior to denaturing process, the protein is of a highquality, and can be used for animal feed.

This summary does not necessarily describe all necessary features of theinvention but that the invention may also reside in a sub-combination ofthe described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows a graphical representation showing the amount of acidrequired to titrate water (dashed line) versus a leachate (solid line).

FIG. 2 shows a graphical representation depicting the amount of acidrequired to titrate water (solid line), a slurry of lignocellulosicfeedstock and water (dashed line), and three leachates which areproduced by leaching lignocellulosic feedstock for 10 minutes (filledsquares), 1 hour (filled diamonds) and 24 hours (filled triangles).

FIG. 3 shows a graphical representation of the effect of leaching timeon the percent alkalinity removal relative to a thoroughly leachedfeedstock.

FIG. 4 shows a graphical representation showing the amount of acidrequired to titrate water (solid line), a leachate derived from leachinglignocellulosic feedstock at 25° C. (filled squares) and a leachatederived from leaching lignocellulosic feedstock at 80° C. (opensquares).

FIG. 5 shows a graphical representation depicting the amount of acidrequired to titrate water (solid line) and four leachates derived fromleaching lignocellulosic feedstock using a mass ratio of water to drylignocellulosic feedstock of about 3:1 (filled squares), 5:1 (filleddiamonds), 7:1 (filled triangles), and 10:1 (filled circles).

FIG. 6 shows several aspects of the present invention. FIG. 6(A) showsan overview of an aspect of the present invention comprising a millingand leaching process. FIG. 6(B) shows a schematic representation of anaspect of the present invention comprising a multistage, countercurrentleaching process.

FIG. 7 shows a graphical representation depicting the amount of acidrequired to titrate unleached lignocellulosic feedstock (solid line),thoroughly leached lignocellulosic feedstock (dashed line), a leachatederived from a single leaching process (filled squares), a leachatederived from a two-stage leaching process (filled diamonds), a leachatederived from a 3 stage leaching process (filled triangles) and aleachate derived from a four stage leaching process (filled circles).

FIG. 8 shows the release of xylose from a lignocellulosic feedstock inthe presence of increasing acid concentration during pretreatment. FIG.8(A) shows a graphical representation depicting total xylose polymerplus monomer release from lignocellulosic feedstock as a function ofacid pretreatment load for an unleached lignocellulosic feedstock (opensquares), a lignocellulosic feedstock treated by leaching at roomtemperature (open triangles), and a lignocellulosic feedstock treated byleaching at 80° C. (open diamonds). FIG. 8(B) shows a graphicalrepresentation depicting xylose monomer release from lignocellulosicfeedstock as a function of acid pretreatment load for an unleachedlignocellulosic feedstock (filled squares), a lignocellulosic feedstocktreated by leaching at room temperature (filled triangles), and alignocellulosic feedstock treated by leaching at 80° C. (filleddiamonds).

DESCRIPTION OF PREFERRED EMBODIMENT

The invention relates to pretreatment processes for the conversion oflignocellulosic feedstocks into sugars. More specifically, the presentinvention relates to pretreatment processes for the conversion oflignocellulosic feedstocks into sugars and the subsequent conversion ofsugars to ethanol.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

According to an embodiment of the present invention there is provided amethod of producing xylose from lignocellulosic feedstock comprising:

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock; and    -   c) pretreating the leached feedstock to produce a composition        comprising xylose, pretreated feedstock, or both xylose and        pretreated feedstock.

The composition comprising xylose and pretreated feedstock, or each ofxylose or the pretreated feedstock may be used for further processing toproduce product of interest, for example but not limited to, ethanol, asdescribed herein.

A non-limiting example of a method to pretreat the lignocellulosicfeedstock, as identified in step c), above, is:

-   -   i) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   ii) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock.        However, any method within the art may be used as required for        the preparation of a pretreated feedstock. Other examples        include, but are not limited to, the methods disclosed in U.S.        Pat. Nos. 6,333,181; 5,198,074; 4,857,145; 4,556,430;4,461,648;        4,237,226; 5,536,325; 5,846,787 (which are incorporated herein        by reference).

By the term “lignocellulosic feedstock” it is meant any type of plantbiomass such as but not limited to non-woody plant biomass, cultivatedcrops such as, but not limited to grasses, for example but not limitedto C4 grasses, such as switch grass, cord grass, rye grass, miscanthus,or a combination thereof, or sugar cane bagasse, soybean stover, cornstover, rice straw, rice hulls, barley straw, corn cobs, wheat straw,oat hulls, corn fiber, recycled wood pulp fiber, sawdust, hardwood,softwood or a combination thereof. Further, the lignocellulosicfeedstock may comprise cellulosic waste material such as, but notlimited to newsprint, cardboard, sawdust and the like. Lignocellulosicfeedstock may comprise one species of fiber or alternatively,lignocellulosic feedstock may comprise a mixture of fibers thatoriginate from different lignocellulosic feedstocks. Further, thelignocellulosic feedstock may comprise fresh lignocellulosic feedstock,partially dried feedstock, fully dried feedstock or a combinationthereof. Preferably, the lignocellulosic feedstock comprises fully driedfeedstock.

Preferably, the lignocellulosic feedstock comprises cellulose in anamount greater than about 20%, more preferably greater than about 30%,still more preferably greater than about 40% (w/w). The lignocellulosicfeedstock may also comprise lignin in an amount greater than about 5%,more preferably in an amount greater than about 10% (w/w). Thelignocellulosic feedstock may also comprise a combined amount ofsucrose, fructose and starch in an amount less than about 20%,preferably less than about 10% (w/w). The weight percentages disclosedabove are relative to the mass of the lignocellulosic feedstock as itexists in its natural state, prior to any processing.

It is preferred that the lignocellulosic feedstock comprise amechanically disrupted feedstock. Mechanical disruption oflignocellulosic feedstock may be performed according to any method knownin the art provided that the method is capable of reducing thelignocellulosic feedstock into particles of an adequate size. Forexample, but not to be considered limiting, mechanical disruption ofstraw preferably results in pieces of straw having a length less thanabout 0.5 inches and an average diameter less than about 2 mm.Preferably, mechanical disruption of lignocellulosic feedstock producesparticles which pass through about 20 mesh, preferably 40 mesh. Withoutwishing to be limiting, mechanical disruption of lignocellulosicfeedstock may be performed by chopping, chipping, grinding, milling,shredding or the like. Preferably, mechanical disruption is performed bymilling, for example, but not limited to, Szego milling, Hammer millingor Wiley milling. However, the method of the present invention alsocontemplates the use of undisrupted lignocellulosic feedstock comprisinga particle size which may pass through about 20 mesh, preferably about40 mesh.

It is preferred that mechanical disruption of lignocellulosic feedstockis performed on feedstock that is substantially dry, that is, thelignocellulosic feedstock comprises less than about 40% moisture,preferably between about 0% and about 40% moisture. More preferably fromabout 0% to about 10% moisture. However, the method of the presentinvention contemplates mechanical disruption of wet lignocellulosicfeedstock or concurrent mechanical disruption and leaching oflignocellulosic feedstock. Furthermore, wet or moist lignocellulosicfeedstock may also be dried prior effecting mechanical disruption of thefeedstock.

In a preferred embodiment the lignocellulosic feedstock comprises afeedstock with a high AX/NSP ratio. AX/NSP is the ratio of arabinan plusxylan to non-starch polysaccharides and can be measured for anyfeedstock based on the analytical procedures described in U.S. Pat. No.6,090,595 which is herein incorporated by reference. AX/NSP iscalculated from EQUATION (1):AX/NSP=(xylan+arabinan)/(xylan+arabinan+cellulose)  (1)wherein the xylan, arabinan, and cellulose contents of the feedstocks ismeasured according to the procedures in EXAMPLE 1. It is preferable thatthe lignocellulosic feedstocks employed in the method of the presentinvention exhibit an AX/NSP ratio of about 0.35 to about 0.45. Mixturesof lignocellulosic feedstocks which exhibit an average AX/NSP ratio ofabout 0.35 to about 0.45 may also be used. However, as would be evidentto someone of skill in the art, a lignocellulosic feedstock or mixtureof feedstocks which comprise an AX/NSP ratio or an average AX/NSP ratiooutside the ranges defined above, may still be employed in the method ofthe present invention.

By the term “leached feedstock” it is meant a lignocellulosic feedstockwhich is or has been in contact with an aqueous solution for a periodgreater than about 2 minutes. The aqueous solution which is, or has beenin contact with the leached feedstock for a period greater than about 2minutes is termed a “leachate”. A leachate may comprise dissolvedsubstances, such as, but not limited to buffering agents' from thelignocellulosic feedstock. Further, the leachate may compriseundissolved substances, such as, but not limited to fine particles ofthe lignocellulosic feedstock.

By leaching the lignocellulosic feedstock the level of compounds thatinterfere with acid pretreatment are reduced. According to the method ofthe present invention, leaching comprises contacting lignocellulosicfeedstock with an aqueous solution for a period between about 2 minutesand about 5 hours, preferably about 2 minutes and about 2 hours, morepreferably about 10 minutes and about 30 minutes. Leaching may beperformed at a temperature between about 4° C. and about 95° C.,preferably between about 20° C. and 80° C. In a preferred embodiment,leaching is performed at about 60° C. to about 80° C., more preferablyat about 70° C. Alternatively, leaching may be performed at atemperature over about 100° C. and under pressure.

Leaching can also recover protein from the feedstock, and this proteinmay be useful as an animal feed. The choice of temperature for theleaching process may therefore involve a balance between the higherleaching efficiency at high temperatures and the protein stability atlower temperature.

In a preferred embodiment, leaching reduces the amount of leachablebuffering agents in the lignocellulosic feedstock from about 50% toabout 100%, more preferably about 70% to about 100%, relative to thetotal leachable buffering agents contained in the lignocelluloicfeedstock prior to the leaching step. The total leachable bufferingagents in a lignocellulosic feedstock may be estimated according toExample 3.

Preferably, the aqueous solution employed in leaching lignocellulosicfeedstock comprises about 0.25 to about 10 times the maximum waterholding capacity per kilogram of dry lignocellulosic feedstock, morepreferably about 1.5 to about 3 times the maximum water holding capacityper kilogram of dry lignocellulosic feedstock. However, leaching may beperformed using an aqueous solution comprising more than about 10 timesthe maximum water holding capacity per kilogram of lignocellulosicfeedstock. The maximum water holding capacity of a lignocellulosicfeedstock may be determined by, for example, measuring the volume ofwater which may be absorbed by a known mass of loosely packedlignocellulosic feedstock until the point at which additional wateradded to the feedstock is free water. This point maybe estimated as thepoint wherein water forms a thin continuous layer over thelignocellulosic feedstock. In determining the maximum water holdingcapacity of a feedstock, it is preferable that the lignocellulosicfeedstock is mechanically disrupted into particles of about the samesize. Further, as would be evident to a person skilled in the art, it ispreferred that the maximum water holding capacity of a feedstock bedetermined on a loosely packed and not tightly packed lignocellulosicfeedstock. The maximum water holding capacity of a lignocellulosicfeedstock may be determined as described in Example 4.

The method of the present invention further contemplates continuousleaching of lignocellulosic feedstock. A continuous leaching process mayemploy a holding tank wherein a continuous feed of lignocellulosicfeedstock and aqueous solution, (forming a slurry) is added to theholding tank and a continuous withdrawal of slurry is made from theholding tank. The contact time, temperature, and mass ratio of aqueoussolution to feedstock are similar to that described previously for batchleaching of lignocellulosic feedstock. A non-limiting example of acontinuous leaching process is shown in FIG. 6(A) and involves atwo-stage countercurrent washing process. The first stage involvespreconditioning of the feedstock, and the second stage comprisesleaching and pressing the feedstock.

In the continuous leaching process as shown in FIG. 6(A), the milledlignocellulosic feedstock (10) is conveyed to a preconditioner (20),which consists of a moving belt (25) onto which low pressure steam (30),for example at about less than 15 psig, is sprayed to wet the fiber. Thepreconditioning heats the feedstock to a temperature of about 80° C.over a period of about 1 minute, that being the time the feedstock is onthe belt. Volatiles in the vapour phase are removed and collected in acondenser (40).

After preconditioning, the feedstock is transferred to a leaching bath(50) where the feedstock remains submerged from about 5 to about 30minutes, preferably about 10 to about 20 minutes. The leaching bath iswhere the initial leaching (leach stage 1) of the preconditionedfeedstock takes place. The leaching bath tank may also be adapted forremoval of sand particles and other heavy debris that may settle to thebottom of the tank. Sand and other debris may be conveyed via conveyor(60) to a sand removal press (70) and discarded in a dumpster (80).

The contents of leach bath (50) are conveyed to a roll press (90) viaconveyor (55), and the feedstock crushed, to facilitate leaching andremoval of liquid, and soluble components, for example but not limitedto protein, salt, sugars and acids, from the solid phase. The pressatefrom the roll press comprising soluble protein, sugars and salts, isrecovered in recovery line (100) and sent for further processing (110),for example, but not limited to protein concentration. The pressate,collected in line (150) from a second press housed within a second leachstage (130), may also be used to wash incoming feedstock in the firststage of the leach, for example at (90). The crushed fiber from the rollpress (90) may be wetted with recycle leachate (120) from a second leachstage (130), and fed to a second press within (130), for example but notlimited to a multi-press, via conveyor (150). The second leach stage(130), involves washing the solids with wash stream (140) and thenpressing the washed solids to separate the solids from the liquid. Theleached solids exit the second leach stage (130) at port (160) and arethen sent for further processing as required using any process as knownwithin the art, for example but not limited to:

-   -   ethanol production as disclosed in U.S. Pat. Nos. 6,333,181 and        5,198,074;    -   paper production as disclosed in U.S. Pat. Nos. 5,198,074 and        4,857,145;    -   biomass hydrolysis U.S. Pat. Nos. 4,556,430, 4,461,648 and        5,846,787;    -   sugar production as disclosed in U.S. Pat. Nos. 4,237,226 and        5,536,325 all of the above are incorporated herein by reference.

As would be evident to someone of skill in the art, lignocellulosicfeedstock may be agitated during leaching, for example, but not wishingto be limiting by stirring or the like. Agitation may also occur throughmovement of the conveyor (55) in leach tank 50 Alternatively, leachingmay be performed without agitation of the lignocellulosic feedstock.

Therefore, the present invention also provides a lignocellulosicfeedstock processing system comprising, a feedstock handling device; apreconditioner capable of receiving the feedstock from the handlingdevice, the preconditioner in communication with a steam source, thesteam source for heating the feedstock, a first transfer device, forconveying the feedstock within, and from, the preconditioner to aleaching bath, a second transfer device for mixing the feedstock withinthe leaching bath and conveying the feedstock from the leaching bath toa press, a third transfer device for conveying pressed feedstock fromthe press to a second leach stage, and multi-press, to produce processedlignocellulosic feedstock.

The aqueous solution employed in the method of the present invention maycomprise, but is not limited to plant water, process water, fresh wateror a combination thereof. However, it is generally preferred that theaqueous solution used in the leaching of the method of the presentinvention comprise an aqueous solution of pH about 3 to about 9,preferably from about pH 6 to about 7, and contain less than about 10g/L of dissolved impurities. The pH of the aqueous solution may beadjusted using any sutaible acid or base, preferably, NaOH or H₂SO₄ Analkaline aqueous solution may be used to help in protein extraction asoutlined above.

It is also contemplated by the method of the present invention thatleaching may be performed in a plurality of stages. For example, but notwishing to be limiting, leaching may comprise from about two to aboutfour leaching stages wherein in each stage the lignocellulosic feedstockis contacted with an aqueous solution under the conditions as definedabove (e.g. see FIG. 6(B)). Following each leaching stage, thelignocellulosic feedstock is removed from the aqueous solution asdiscussed below.

The step of removing the leachate from the leached feedstock may beperformed according to any process known in the art. For example, butnot wishing to be limiting, the leachate may be removed from the leachedfeedstock by draining, pressing, filtering, screening, centrifuging or acombination thereof. Alternatively, but without wishing to be limiting,the leachate may be removed from the leached feedstock by rinsing orwashing the leached feedstock using a washer.

As would be evident to someone of skill in the art, the step of removingthe leachate from the leached feedstock need not result in completeremoval of all aqueous solution from the leached feedstock. However, itis preferred that between about 20% and about 100%, preferably about 50%and about 100% of the leachate be removed from the leached feedstock.

The leachate produced in the method of the present invention may be usedin processes related to or unrelated to ethanol production.Alternatively, the leachate may be discarded. Further, the leachate maybe examined during or after leaching of lignocellulosic feedstock todetermine the quantity of soluble components and contaminants removedfrom the lignocellulosic feedstock. The soluble components may include,but are not limited to buffering agents and the like. Therefore, themethod of the present invention further contemplates sampling theleachate during leaching, after leaching or both before and afterleaching to determine the extent of leaching of the lignocellulosicfeedstock. Thus, it is possible to optimize leaching parameters such as,but not limited to, the leaching times of a lignocellulosic feedstock,or the number of leaching stages required for a lignocellulosicfeedstock. Further, it is possible to monitor the extent of leachingwhen practicing the method of the present invention.

Following the step of removing the leachate from the leached feedstock,the feedstock may be processed using any desired method for theproduction of ethanol. Non-limiting examples of such methods includethose described in U.S. Pat. Nos. 6,333,181; 5,198,074; 4,857,145;4,556,430; 4,461,648; 4,237,266,5,221,537,5,536,325; 5,628,830;5,846,787 (which are incorporated herein by reference).

A preferred method to further process the leached feedstock comprisesthe step of acidifying the leached feedstock to a pH between about 0.5and about 8, preferably, a pH of about 0.5 to about 3 to produce anacidified feedstock. The leached feedstock may be acidified using anyacid known in the art, but is preferably acidified using sulfuric acid,nitric acid or hydrochloric acid. In a preferred embodiment the acid issulfuric acid.

Reacting acidified feedstock under conditions which disrupt fiberstructure as contemplated in the method of the present invention, may beperformed according to any method known in the art, for example, but notlimited to pretreatment by steam explosion as described in U.S. Pat Nos.4,461,648, and 4,237,226 which are herein incorporated by reference.Further, any parameters used in the prior art to effect steam explosionpretreatments, such as, but not limited to those described in Foody, etal, Final Report, Optimization of steam Explosion Pretreatment, U.S.DEPARTMENT OF ENERGY REPORT ET230501 (April 1980, which is hereinincorporated by reference) maybe used in the method of the presentinvention.

Preferably, the step of reacting the acidified feedstock is performedusing a temperature between about 100° C. to about 220° C. at about pH0.5 to about 2.5 for about 5 seconds to about 60 minutes. It isunderstood by those skilled in the art that the feedstock temperature isthat of the feedstock itself, which might differ from the temperaturemeasured outside the reaction chamber. Devices used to carry out thispretreatment include, but are not limited to sealed batch reactors,continuous extruders and steam guns.

It is also within the scope of the present invention that a two-stagepretreatment process may be used, whereby the first stage improves thecellulose hydrolysis somewhat while solubilizing primarily thehemicellulose but little cellulose. The second stage then completes afull pretreatment. In this embodiment, the first stage reaction is runat a temperature of less than about 180° C. while the second stagereaction is run at a temperature of greater than about 180° C.Preferably, the first stage of the reaction is carried out at atemperature of about 60° C. to about 140° C. for 0.25 to 24 hours at pH0.5 to 2.5. More preferably, the first stage of pretreatment is carriedout at a temperature of 100° C. to 130° C. for 0.5 to 3 hours at pH 0.5to 2.5. While the second stage of reaction may be carried out at atemperature of 180° C. to 270° C., at pH 0.5 to 2.5 for a period of 5seconds to 120 seconds. The two-stage pretreatment provides separaterecovery of the hemicellulose for downstream processing.

Reacting the acidified feedstock as contemplated by the method of thepresent invention, produces a composition comprising xylose and apretreated feedstock. Preferably, as a result of the methods asdescribed herein, the composition comprises between about 150 to about260 mg of xylose per gram of dry lignocellulosic feedstock. As will beevident to someone of skill in the art, the composition may comprisesugars other than xylose and a variety of other components. The xylosemay be purified from the composition.

The composition or pretreated feedstock derived from practicing themethod of the present invention may be treated With cellulase enzymes tohydrolyse cellulose to glucose using methods as would be known to one ofskill in the art. A cellulase enzyme treatment may be performed bymixing the pretreated feedstock or composition derived from the methodof the present invention with water and titrating the mixture to a pH ofabout 5 to achieve a slurry of 5% to 10% by weight cellulose. Cellulaseenzymes are then added to the mixture. Typically, the hydrolysis is runfor about 24 to about 200 hours at about 50° C. At the end of thehydrolysis, glucose and other water soluble sugars remain dissolved insolution, while unconverted cellulose, lignin, and other insolubleportions of the substrate remain in suspension or precipitate fromsolution. A syrup comprising glucose and other dissolved sugars andsolutes may be recovered by filtering the hydrolysis slurry. Washing ofthe fiber solids may also be performed to increase the yield of glucoseand other sugars.

It is also contemplated that glucose produced by the hydrolysis ofcellulose from the pretreated feedstock may be fermented to ethanol.Fermentation of glucose and other sugars to ethanol may be performed byconventional processes known to those skilled in the art, and may beeffected by a variety of microorganisms including yeast and bacteria orgenetically modified microorganisms, for example, but not limited thosedescribed in WO95/13362, WO97/42307 and the article entitled Alcoholproduction from Cellulosic Biomass: The Iogen Process in: The AlcoholTextbook.(2000, Nottingham University Press) which are hereinincorporated by reference).

It is also contemplated that both glucose and xylose produced in themethod of the present of the present invention may be fermented toethanol. Alternatively, xylose and glucose maybe fermented to ethanolindependently. Ethanol production and recovery are performed bywell-established processes used in the alcohol industry.

Thus, the present invention, provides a method of producing ethanol froma lignocellulosic feedstock comprising,

-   -   a) leaching the lignocellulosic feedstock by contacting the        feedstock with at least one aqueous solution for a period        greater than about 2 minutes to produce a leached feedstock and        a leachate;    -   b) removing the leachate from the leached feedstock;    -   c) acidifying the leached feedstock to a pH between about 0.5        and about 3 to produce an acidified feedstock, and;    -   d) reacting the acidified feedstock under conditions which        disrupt fiber structure and hydrolyze a portion of hemicellulose        and cellulose of the acidified feedstock, to produce a        composition comprising xylose and a pretreated feedstock;    -   e) treating the composition comprising xylose and pretreated        feedstock with cellulase under conditions which hydrolyse        cellulose in the pretreated feedstock to glucose, producing a        sugar solution comprising xylose and glucose, and;    -   f) either:        -   i) fermenting the sugar solution to ethanol;        -   ii) treating the composition with a microrganism under            conditions which permit fermentation of the xylose in the            composition to ethanol; or        -   iii) separating the pretreated feedstock form the            composition, and treating the pretreated feedstock with            cellulase under conditions which hydrolyse cellulose in the            pretreated feedstock to glucose, producing a sugar solution            comprising glucose, and fermenting the sugar solution to            ethanol.            Thus, it is contemplated that xylose produced in the            pretreatment of lignocellulosic feedstock may be fermented            in combination with glucose, or separate from glucose            produced by cellulose hydrolysis of the pretreated            feedstock.

It is also contemplated that the glucose may be fermented to lactic acidor converted to other useful products such as, but not limited tosorbitol, acetic acid, citric acid, ascorbic acid, propanediol,butanediol, acetone, butanol, or a combination thereof.

Leaching of lignocellulosic feedstock as performed in the method of thepresent invention reduces the amount of acid required in a hydrolysisprocess compared to lignocellulosic feedstock which has not beensubjected to leaching. Referring now to FIG. 1, there is shown theamount of acid required to titrate water to a desired pH (dashed line)relative to the amount of acid required to titrate a leachate solution(solid line) comprising an aqueous solution which has been in contactwith a lignocellulosic feedstock according to the method of the presentinvention. The data from FIG. 1 indicate that lignocellosic feedstockscomprise buffering agents may be removed from the feedstock by leachingwith aqueous solution. Removing all or a portion of the buffering agentsfrom a lignocellulosic feedstock reduces the amount of acid that must beused in a dilute acid prehydrolysis process such as, but not limited tosteam explosion.

Referring now to FIG. 2, there is shown the amount of acid required totitrate water, an unleached lignocellulosic feedstock slurry, or threeleachates which have been used to contact three lignocellulosicfeedstocks for a period of 10 minutes, 1 hour or 24 hours, respectively.The results shown in FIG. 2 suggest that leaching lignocellulosicfeedstock for a period between about 10 minutes and about 24 hours iscapable of removing buffering agents from the feedstock so as to reducethe amount of acid required to titrate the pH of the leachedlignocellulosic feedstock.

Referring now to FIG. 3, there is shown the percent alkalinity removalas a function of leaching time for a lignocellulosic feedstock relativeto a thoroughly leached feedstock prepared according to Example 4. Theresults shown in FIG. 3 suggest that a leaching time of about 2 minutesis capable of leaching about 48% of the total leachable buffering agentsfrom the lignocellulosic feedstock and a time of about 60 minutesleaches about 85% of the total leachable buffering agents.

Referring now to FIG. 4 there is shown the effect of temperature onleaching of lignocellulosic feedstock. These data indicate that leachingof lignocellulosic feedstock may be performed under a wide range oftemperatures. Specifically, lignocellulosic feedstock treated byleaching at temperatures between about 25° C. and about 80° C. produceleachates comprising buffering agents. Removal of buffering agents fromlignocellulosic feedstocks during leaching produces leached feedstockwhich requires less acid load to titrate the feedstock to an acidic pHsuitable for dilute acid prehydrolysis processes such as, but notlimited to, steam explosion. While the results shown in FIG. 4 suggestthat leaching temperatures of between about 25° C. and about 80° C. maybe used in the method of the present invention, other temperaturesoutside this range may also be used in the method of the presentinvention.

Referring now to FIG. 5, there is shown the amount of acid required totitrate leachates, wherein the mass ratio of aqueous solution (leachate)to dry lignocellulosic feedstock to is about 3:1, 5:1, 7:1 and 10:1. Theresults depicted in FIG. 5 are corrected for volume and suggest that awide range of leachate to feedstock ratios may remove similar amounts ofbuffer agents from lignocellulosic feedstocks. The minimum ratio ofaqueous solution to lignocellulosic feedstock corresponds to an amountabout equal to or greater than the maximum water holding capacity of thefeedstock.

The results depicted in FIGS. 1-5 are based on a single leaching oflignocellulosic feedstock. However, the method of the present inventionalso contemplates multiple teachings of lignocellulosic feedstock. Forexample, but not to be considered limiting, lignocellulosic feedstockmay be subjected to multiple leaching stages, wherein after eachleaching, the leachate is removed from the leached feedstock asdescribed previously herein. In embodiments of the method of the presentinvention wherein multiple teachings of the lignocellulosic material areperformed, preferably the method comprises between about 2 and about 4leachings of the lignocellulosic feedstock. Without wishing to be boundby theory, multiple teachings may allow the volume of aqueous solutionper mass of lignocellulosic feedstock to be reduced in practicing themethod of the present invention.

The method of the present invention further contemplates multipleteachings of lignocellulosic feedstock wherein the multiple teachingsare performed in a countercurrent fashion. Referring now to FIG. 6,there is shown a 1-4 stage countercurrent leaching process which may beemployed in the method of the present invention. FIG. 6 is provided asan example and is not intended to be limiting in any manner. As shown inFIG. 6, a first lignocellulosic feedstock is leached with water in afirst leaching stage to produce a first leachate and a first leachedfeedstock. Following the appropriate leaching time, the first leachateis separated from the first leached feedstock. This represents a onestage leaching of lignocellulosic feedstock.

As further shown in FIG. 6, the first leachate or an aliquot thereof maybe used to leach a second lignocellulosic feedstock thereby producing asecond leachate and a second leached feedstock. The second leachate, oran aliquot thereof, may be used to leach a third lignocellulosicfeedstock. Also shown in FIG. 6, the second leached feedstock is furtherleached with water in a second leaching stage. The leachate produced inthis second leaching may be used to leach a further lignocellulosicfeedstock which has been previously leached or unleached. The entireprocess maybe repeated for any number of stages. However, in embodimentsof the present invention which employ multiple teachings oflignocellulosic feedstock, it is preferred that 2 to 4 leaching stagesare used. Further, although FIG. 6 represents a countercurrent leachingprocess, wherein a leachate from a first leaching is employed in theleaching of a second lignocellulosic feedstock, it is also contemplatedthat each leaching may be performed with an aqueous solution that wasnot previously used to leach lignocellulosic feedstock.

In embodiments of the method of the present invention wherein multipleteachings of lignocellulosic feedstock are performed in a countercurrentmanner, it is preferred that a subsequent leaching of a lignocellulosicfeedstock employ an aqueous solution containing less impurities than aprevious leaching of the same lignocellulosic feedstock, as would beknown to someone of skill in the art. More preferably, the last leachingof a multiple leaching process comprises substantially pure water, thatis water that has not been previously used to leach a feedstock.

Referring now to FIG. 7, there is shown the amount of acid required totitrate an unleached lignocellulosic feedstock, four leachedlignocellulosic feedstocks comprising between 1 and 4 teachings and athoroughly leached lignocellulosic feedstock The results shown in FIG. 7suggest that multiple teachings of lignocellulosic feedstock may be usedto remove buffering agents from the feedstock thereby reducing the acidload required for dilute acid prehydrolysis processes, for example, butnot limited to steam explosion. Thus, the method of the presentinvention contemplates multiple leachings of lignocellulosic feedstock.In a preferred embodiment, the present invention contemplates from about1 to about 5 Teachings of lignocellulosic feedstock wherein the volumeof aqueous solution is about 3 to about 10 times the volume of themaximum water holding capacity per kilogram of the lignocellulosicfeedstock, more preferably about 7 times the maximum water holdingcapacity of the lignocellulosic feedstock.

The results depicted in FIGS. 1-7 suggest that leaching oflignocellulosic feedstock may reduce the amount of acid required totitrate the pH of the leached feedstock to a value suitable for adilute-acid hydrolysis. Thus, the present invention further contemplatesa method of reducing the amount of acid required to effect dilute acidhydrolysis treatment of lignocellulosic feedstock. Further, leaching oflignocellulosic feedstock may be used to reduce the amount of acidrequired in other processes such as, but not limited to those processesemploying acid to depolymerize cellulose to glucose. Thus, the presentinvention contemplates a method of reducing the amount of acid requiredto effect acid depolymerization of cellulose to glucose.

Leaching of lignocellulosic feedstock is capable of removing bufferingagents from the feedstock. Thus, leaching lignocellulosic feedstock maybe employed to reduce the amount of base required to titrate the pH ofthe lignocelluloic feedstock to an appropriate pH for other processes,such as but not limited to ammonia pretreatment of lignocellulosicfeedstock. The present invention contemplates a method of reducing theamount of base required to effect an ammonia pretreatment oflignocellulosic feedstock.

Xylose Production

A drawback of dilute acid prehydrolysis processes known in the art isthat these processes destroy xylose during treatment. The destruction ofxylose during dilute acid prehydrolysis reduces the ethanol yield thatmay be obtained from the feedstock.

Referring now to FIG. 8(A), there is shown the amount of xylose polymerreleased from lignocellulosic feedstock as a function of acid load in adilute-acid pretreatment process. As shown in FIG. 8(A), leaching oflignocellulosic feedstock prior to dilute acid pretreatment results in ahigher amount of xylose polymer release from lignocellulosic feedstockthan does unleached lignocellulosic feedstock treated in an identicalmanner. Furthermore, the amount of xylose released from pretreatedfeedstock is greater than the unleached feedstock for any given amountsof acid used during pretreatment, and the amount of acid required toeffect xylose release is much less than that of the unleached feedstock.

Referring now to FIG. 8(B), there is shown the amount of xylose monomerreleased from lignocellulosic feedstock as a function of acid load in adilute-acid pretreatment process. As shown in FIG. 8(B), leaching oflignocellulosic feedstock prior to dilute acid pretreatment results in ahigher amount of xylose monomer release from lignocellulosic feedstockthan does unleached lignocellulosic feedstock treated in an identicalmanner. Again, the amount of xylose released from pretreated feedstockis greater than the unleached feedstock for any given amount of acidused during pretreatment. The amount of acid required to effect xyloserelease is also much less than that of the unleached feedstock.

Therefore, the method of the present invention increases the amount ofxylose released from lignocellulosic feedstocks following dilute acidprehydrolysis processes and increases ethanol yield when the samefeedstock is treated with microorganisms, cellulase enzyme, or both, andsubsequently fermented to ethanol.

The above description is not intended to limit the claimed invention inany manner, Furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposed only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLES Example 1

MEASUREMENT OF AX/NSP IN FEEDSTOCKS

The ratio of arabinan plus xylan to total non-starch polysaccharides ofa given feedstock is determined based on a compositional analysis of thefeedstocks. This analysis is performed, as follows:

Feedstocks examined include barley straw, wheat straw, wheat chaff, oathulls, switch grass, corn stover, maple wood, pine wood, and threevarieties of corn cobs. All are obtained locally in Ottawa, Ontarioexcept the oat hulls, which are from Quaker Oats in Peterborough,Ontario. The feedstocks are coarsely ground in a Waring blender and thenmilled through a #20 mesh screen using a Wiley mill. The feedstocks arestored at ambient temperature in sealed bags until the time of use. Themoisture content of small samples is 5% to 10% and is determined bydrying at 100° C.

Approximately 0.2 grams of sample is weighed into test tubes, eachcontaining 2.5 ml of 72% sulfuric acid. The tubes are vortex mixed,capped, and placed in a 40° C. water bath for 30 minutes, with vigorousvortex mixing every 10 minutes. After the 30 minute incubation, the tubecontents are transferred into preweighed 250 ml flasks containing 97.5ml deionized water, which reduced the acid content to 1.8%. Each flaskis transferred to a steam autoclave and is maintained at 121° C. for 1hour. The flask is weighed after autoclaving

The concentrations of glucose, xylose, and arabinose present in thefiltrates are measured by using a Dionex Pulse-Amperometric HPLC. Thesemeasurements are then related to the weight of the initial sample offeedstock present and expressed as glucan, xylan, and arabinan contents,respectively, of the feedstock, with small adjustments to take intoaccount (1) the water of hydration to make the monomers from polymersand (2) the amount of material destroyed by the concentrated acid, whichis measured by taking pure cellulose, xylose, and arabinose controlsthrough the procedure. The determination is performed in triplicate andthe average value is reported.

The cellulose content is determined by subtracting the starch contentfrom the total glucan. The starch content is determined by adding 1 gramof Wiley-milled feedstock to a 250 ml flask containing 20 ml ofdeionized water, 0.2 ml of 91.7 g/L CaCl₂.2H₂O stock solution, and 50microliters of a 1:100 solution of Sigma Alpha Amylase #A3403 indeionized water. Each flask is adjusted to pH 6.4 to 6.6 using dilutesodium hydroxide, then incubated in a boiling water bath for one hour.The flasks are incubated for 30 minutes in a steam autoclave at 121° C.after the addition of a second 50 μl dose of amylase. Finally, the flaskis incubated for another 60 minutes in the boiling water bath with athird 50 μl dose of amylase. The flasks are then cooled to ambienttemperature and adjusted to pH 4.2 to 4.4 using dilute hydrochloricacid. A 0.5 ml aliquot of Novo Spritamylase stock solution is added; thestock solution consisted of 3 grams of enzyme solution in 100 mldeionized water. The flasks are shaken at 50° C. for 20 hours with 150RPM agitation. The flasks are then cooled and the contents are filteredover glass microfiber filter paper. The glucose concentration is thenmeasured on a Yellow Springs Instrument (YSI) glucose analyzer and usedto determine the starch concentration of the feedstock, taking intoaccount the water necessary to hydrolyse the starch.

The protein and ash content of the feedstocks are determined by standardKjeldahl nitrogen and ash oven methods.

The lignin content of the samples is determined by measuring the amountof insoluble solids remaining after the sulfuric acid treatment of thefeedstocks, then subtracting the amount of ash present.

The results of these measurements are shown in TABLE 1. The materialrecovered is between 849 and 1018 mg per gram of original solids (mg/g).This corresponds to 84.9%, by weight, to 101.8% of the startingmaterial, which is typical mass balance closure in these systems.

TABLE 1 COMPOSITION OF THE FEEDSTOCKS Measured composition (mg/g)Feedstock Glucan Starch Xylan Arabinan Lignin Ash Protein Total BarleyStraw 426 19.6 161 28 168  82 64 929 Wheat Straw 349 12   167 18 204  8364 885 Wheat chaff 405 14.4 200 36 160 121 33 955 Switch grass 331  3.4212 24 183  48 54 852 Corn stover 370  2.3 189 22 127  60 81 849 Maplewood 504 4  150  5 276  6  6 947 Pine wood 649 1   33 14 320  0  2 1018 Corn cobs (red) 436 34   253 38 ND (2) ND ND ND Corn cobs (white) 43928   250 38 ND ND ND ND Corn cobs (Indian) 438  8.5 240 36 ND ND ND NDOat Hulls 436 90   187 26 170  44 38 901 Soybean Stover 377 ND 120 13 NDND ND ND (1) Total = Glucan + Xylan + Arabinan + Lignin + Ash + Protein(2) ND = Not determined

The AX/NSP content of the feedstocks is shown in TABLE 2. Of the 12feedstocks analyzed, eight have AX/NSP of greater than about 0.35. Theseinclude the samples of wheat straw, wheat chaff, switch grass, cornstover, oat hulls and corn.

TABLE 2 AX/NSP COMPOSITION OF THE FEEDSTOCKS Cellulose Feed-stock (mg/g)(1) AX (mg/g) (2) NSP (mg/g) (3) AX/NSP Barley Straw 407 189 596 0.317Wheat Straw 337 185 522 0.354 Wheat chaff 391 236 627 0.376 Switch grass328 236 564 0.418 Corn stover 347 211 558 0.378 Maple wood 500 155 6550.237 Pine wood 648 47 695 0.068 Corn cobs 402 291 693 0.42 (red) Corncobs 411 288 699 0.412 (white) Corn cobs 429 276 705 0.391 (Indian) OatHulls 346 213 559 0.381 Soybean 377 133 510 0.26 Stover (1) Cellulose =Glucan − Starch (2) AX = Xylan + Arabinan (3) NSP = Xylan + Arabinan +Cellulose

Example 2 Leaching and Dilute Acid Pretreatment of LignocellulosicFeedstock

A. Leaching

Wheat straw is ground using a Wiley mill to produce particles of a sizeless than 20 mesh. The milled lignocellulosic feedstock is added towater at 25° C. or 80° C. for leaching. For comparative purposes anequivalent amount of Wiley milled straw is set aside as a control andprocessed without leaching. Leaching of the lignocellulosic feedstock isperformed for 60 minutes using a mass ratio of water to lignocellulosicfeedstock of 10:1. Following leaching, the leached lignocellulosicmaterial is filtered and pressed to 50% solids content.

B. Dilute Acid Hydrolysis of Leached Feedstock

The leached lignocellulosic material (1.0 grams, total fiber) is placedin a 10 mL stainless steel reactor. A volume of 5% sulfuric acid isadded to the leached feedstock to obtain a ratio of acid to leachedfeedstock in the range of 0.5% to 6.3% weight/weight. The reactor issealed and placed in a 200° C. oil bath for 2 minutes. Following the 5minute incubation the reactor is placed in cold water to quench thereaction.

C. Determination of Xylose Concentration

The xylose concentration of the treated feedstock is measured byfiltering an aliquot and hydrolysing the sample at 120° C. with 2%sulfuric acid. The xylose concentration of the hydrolysate is measuredby HPLC and is expressed in milligrams of xylose per gram oflignocellulosic feedstock.

The results obtained for xylose release from lignocellulosic feedstocktreated according to the method of the present invention are shown inFIGS. 8A and 8B. The highest xylose yield obtained with 25° C. leachingwas about 170 mg/g, representing an improvement of about 20% overlignocellulosic biomass that is not subjected to leaching prior topretreatment. An acid load of about 4.5% on the feedstock is required toreach this yield. An acid load of about 2.5% is required to produce 140mg/g xylose according to the method of the present invention. Withoutleaching, a similar process requires about 6.5% acid load performedunder the conditions described above.

D. Leaching Time

Experiments in which leaching time is varied are performed in a similarmanner as those described above, except that the leaching times are 2minutes, 10 minutes, 1 hour and 24 hours. The results of varying theleaching time are shown in FIGS. 2 and 3.

E. Leaching Temperature

Experiments in which the leach temperature is varied are performed in asimilar manner as those described above, except that the leachingtemperatures are 25° C. and 80° C. The results of varying the leachingtemperature are shown in FIG. 4.

F. Ratio of Lignocellulosic Feedstock to Water Ratio

Experiments in which the mass ratio of water (leachate) tolignocellulosic feedstock (leachate) is varied are performed in asimilar manner as those described above, except that the mass ratio ofwater to lignocellulosic feedstock to water is 3:1, 5:1, 7:1 and 10:1.The results of these Teachings are shown in FIG. 5

G. Multiple Leaching Stages

The leaching stage is performed in a similar manner with a mass ratio ofwater to lignocellulosic feedstock of about 5:1. Leaching is performedfor 10 minutes at 25° C., followed by pressing the leachedlignocellulosic feedstock to 50% solids (mass ratio of water tofeedstock of about 0.5:1. Additional leaching stages are repeated aspreviously described. The results of 0-4 leaching stages are shown inFIG. 7.

Example 3

Preparation of a Thoroughly Leached Feedstock.

25 grams of lignocellulosic feedstock is milled to a particle sizecapable of passing through 20 mesh. Leaching of the lignocellulosicfeedstock is performed by combining the lignocellulosic feedstock with10 times the volume of its maximum water carrying capacity and theslurry is stirred at 25° C. for 24 hours. After the incubation period,the leachate is removed from the leached feedstock and the feedstock isrinsed with about 2L of a water and pressed to a solids content of about50%. The pressed, leached feedstock is a thoroughly leached feedstock.

The total leachable buffering agents of the feedstock is determined bytitrating the leachate plus rinse water to pH 3 with 0.1N: H₂SO₄. Thevolume of titrant used is expressed as kg H₂SO₄ per tonne of drylignocellulosic feedstock. This is a measure of the total leachablebuffering agents of the feedstock.

Example 4

Maximum Water Holding Capacity of Feedstock

A sample of about 25 grams of lignocellulosic feedstock with known dryweight and moisture content is mechanically disrupted into particles ofabout equal size and that pass through 20 mesh. The lignocellulosicfeedstock is sifted into a container, allowing the feedstock to packunder the influence of gravity. Water is added gradually to a known massof feedstock in a test tube until the point at which additional wateradded is free water. This point is estimated as the point wherein waterforms a thin continuous layer over the lignocellulosic feedstock. Thetest tube is tipped to remove free water. The wet feedstock is thenweighed to determine the amount of water and feedstock present. The massof water in the particles at this point is the maximum water holdingcapacity of the lignocellulosic feedstock for that particular amount andtype of feedstock. The maximum water holding capacity per mass oflignocellulosic feedstock may be measured by dividing the mass of waterrequired to reach the point wherein the addition of further waterresults in free water over the lignocellulosic feedstock, by the mass ofthe lignocellulosic feedstock. Thus, leaching lignocellulosic feedstockusing 10 times the maximum water holding capacity per kilogram of drylignocellulosic feedstock means that the mass of aqueous solution isequal to 10 times the product of the mass of the dry lignocellulosicfeedstock in kilograms and the maximum water holding capacity perkilogram of feedstock.

Example 5

Method of Producing Ethanol

A 0.28 gram dry sample of Wiley-milled oat hull lignocellulosicfeedstock is leached for 10 minutes in 2 ml of tap water. The leachateis removed and the lignocellulosic feedstock is pressed to about 50%(w/v) solids content and subsequently, the leached feedstock is placedin 7 grams of 1% sulfuric acid (pH 0.6 to 0.9) in a sealed stainlesssteel “bomb” reactor. The capacity of the bomb reactor is 9 ml. The bombreactor is placed in a preheated 290° C. oil bath for 50 seconds, andthen cooled under tap water. The leached feedstock is now referred to asa pretreated feedstock Thermocouple measurements showed that thetemperature in the interior of the bomb reached 260° C. by the end ofthe heating period. The average equivalent temperature was 235° C.

The pretreated feedstock is rinsed with tap water, and thenvacuum-filtered over glass microfiber filter paper. The filter cakes arewashed with tap water and air dried.

The pretreated feedstock is subjected to hydrolysis by cellulase asfollows. A sample of the pretreated feedstock corresponding to 0.05grams of cellulose is added to a 25 ml flask with 4.9 grams of 0.05 Msodium citrate buffer, pH 4.8. Iogen Cellulase (140 Filter paper units(FPU)/ml) and Novozym 188 beta-glucosidase (1440 BGU/ml) are added tothe flask in an amount corresponding to 9 FPU/gram cellulose and 125BGU/gram cellulose.

The flask is placed on an Orbit gyrotory shaker at 50° C. and shaken for20 hours at 250 RPM. At the end of this period, the contents of theflask is filtered over glass microfiber filter paper. The filtrate,which contains glucose and other sugars is pH adjusted, inoculated witha precultured mid log phase (about 400 Klett units) S. cerevisiae cellculture and the mixture is fermented anerobically at 30° C. with shakingfor 48 hours. After 48 hours of incubation, the mixture is centrifugedto separate yeast from the medium containing ethanol. The ethanol may bepurified by distillation.

Using the methods described herein for biomass processing and ethanolproduction, yields of about 300-340 liter ethanol per metric ton drybiomass have been produced.

Example 5 teaches washing of pretreated feedstock prior to convertingcellulose to glucose and fermenting glucose to ethanol. However, it isalso contemplated that the entire composition treated in the bombreactor may be pH adjusted, treated with cellulase to convert celluloseto glucose and fermented to ethanol. In such an embodiment, xylose isnot removed from the pretreated feedstock by washing. In still anotherembodiment, xylose may be removed from the pretreated feedstock andfermented independently of glucose produced from cellulose hydrolysis ofthe pretreated feedstock.

As will be evident to someone of skill in the art, fermentation ofsugars such as, but not limited to glucose and xylose may be performedusing a variety of microorganisms, such as, but not limited to yeast,bacteria or a combination thereof. Further, the organisms maybegenetically modified organisms. Also, as is evident to someone of skillin the art, there exists other fermentation conditions which may beemployed in the method of the present invention.

Also, as is evident to someone of skill in the art, ethanol may berecovered and purified from cultures medium by an method know in theart. These processes are fully contemplated in embodiments of the methodof the invention.

All References are herein Incorporated by Reference.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

1. A method of producing xylose comprising, in this order: a) leaching alignocellulosic feedstock comprising greater than about 20% (w/w)cellulose, said leaching comprising contacting said feedstock with atleast one aqueous solution of about pH 6 to about pH 9 for between about2 minutes and about 5 hours, at a temperature of about 25° C. to about80° C. to produce a leached feedstock and a leachate; b) removingleachate from said leached feedstock; and c) treating said leachedfeedstock with an aqueous solution at a temperature of at least about100° C. and at a pH of about 0.5 to about 2.5 to produce a compositioncomprising xylose and a treated feedstock.
 2. The method of claim 1,wherein said lignocellulosic feedstock is selected from the groupconsisting of sugar beet bagasse, soybean stover, corn stover, ricestraw, rice hulls, barley straw, corn cobs, wheat straw, wheat chaff,oat hulls, corn fiber, wood fiber, switch grass, cord grass, rye grass,miscanthus, and a combination thereof.
 3. The method of claim 2, whereinsaid lignocellulosic feedstock comprises mechanically disruptedfeedstock.
 4. The method of claim 3, wherein said mechanically disruptedfeedstock is capable of passing through about 20 mesh.
 5. The method ofclaim 2, wherein said lignocellulosic feedstock has an AX/NSP ratio ofabout 0.35 to about 0.45.
 6. The method of claim 5, wherein saidfeedstock comprises wheat straw, wheat chaff, switch grass, corn stover,corn cobs, oat hulls, or a combination thereof.
 7. The method of claim3, wherein said disrupted feedstock is produced by shredding, milling,chopping, chipping, grinding or a combination thereof.
 8. The method ofclaim 7, wherein said milling comprises Szego milling, Hammer milling,Wiley milling, or a combination thereof.
 9. The method of claim 1,wherein said leaching is performed for a period between about 2 minutesand about 2 hours.
 10. The method of claim 1, wherein the amount ofaqueous solution of step (a) added during said leaching is between about0.25 to about 10 times the maximum water holding capacity of saidlignocellulosic feedstock, wherein the maximum water holding capacity ismeasured by: (i) mechanically disrupting a sample of the lignocellulosicfeedstock; (ii) adding water to a known dry mass of the mechanicallydisrupted feedstock until the point at which additional water added isfree water; (iii) determining the mass of water required to reach thepoint at which additional water added is free water; and (iv) dividingthe mass of water determined in step (iii) by the known dry mass ofmechanically disrupted feedstock to obtain a value representing themaximum water holding capacity.
 11. The method of claim 10, wherein theamount of aqueous solution added during said leaching is about 1.5 to 3times the maximum water holding capacity of said lignocellulosicfeedstock.
 12. The method of claim 1, wherein said at least one aqueoussolution of step (a) comprises water, mill water, process water, aleachate or a combination thereof.
 13. The method of claim 1, whereinsaid pH of the at least one aqueous solution of step (a) is adjustedusing NaOH, H₂SO₄, or a combination thereof.
 14. The method of claim 1,wherein said leaching comprises a plurality of leaching stages.
 15. Themethod of claim 14, wherein said leaching stages comprise 2 to 4leaching stages.
 16. The method of claim 14, wherein said plurality ofleaching stages are performed such that leachate removed from adownstream leaching stage is added to an upstream leaching stage. 17.The method of claim 1, wherein said leaching removes between about 50%to about 100% of the total leachable buffering agents from thelignocellulosic feedstock.
 18. The method of claim 1, wherein saidleaching removes between about 70% to about 100% of the total leachablebuffering agents from the lignocellulosic feedstock.
 19. The method ofclaim 18, wherein said leaching removes between about 90% to about 100%of the total leachable buffering agents from the lignocellulosicfeedstock.
 20. The method of claim 1, wherein the treating of step (c)is performed using an acid selected from the group consisting ofsulfuric acid, nitric acid and hydrochloric acid.
 21. The method ofclaim 20, wherein said acid is sulfuric acid.
 22. The method of claim 1,wherein said leachate is sampled during leaching, following leaching orboth, and wherein conditions of the leaching are adjusted accordingly.23. The method of claim 1, wherein the xylose is a xylose monomer. 24.The method of claim 1, wherein said composition produced from saidmethod comprises a xylose concentration in the range of about 150 mg toabout 260 mg of xylose per gram of feedstock.
 25. The method of claim 1,wherein said treated feedstock has increased accessibility to beingdigested with cellulase enzymes.
 26. The method of claim 1, wherein saidmethod further comprises cellulase treatment of said treated feedstock.27. The method of claim 10, wherein said method further comprisescellulase treatment of said treated feedstock.
 28. The method of claim1, wherein said at least one aqueous solution of step (a) comprises lessthan about 10 g/1 impurities.
 29. The method of claim 1, wherein saidlignocellulosic feedstock comprises cellulose in an amount greater thanabout 30% (w/w).
 30. The method of claim 1, wherein said lignocellulosicfeedstock comprises a lignin content in an amount greater than about 5%(w/w).
 31. The method of claim 1, wherein said lignocellulosic feedstockcomprises a combined sugar content of sucrose, fructose and starch ofless than about 20% (w/w).
 32. The method of claim 1, wherein saidlignocellulosic feedstock comprises oat hulls, wheat straw, switchgrass, or a combination thereof.
 33. The method of producing xyloseaccording to claim 3, wherein said contacting with said at least oneaqueous solution is for a period of about 2 minutes to about 2 hoursusing a volume of the at least one aqueous solution which is betweenabout 0.25 and about 10 times the maximum water holding capacity of saiddisrupted lignocellulosic feedstock per kilogram of dried feedstock. 34.The method of claim 1, further comprising, prior to said leaching step(a), heating said lignocellulosic feedstock to about 80° C.
 35. Themethod of claim 1, wherein in said leaching step (a), thelignocellulosic feedstock is incubated within a leaching bath and debrisis removed.
 36. The method of claim 35, wherein in said leaching step(a), said lignocellulosic feedstock is incubated from about 5 to about30 minutes.
 37. The method of claim 1, wherein said step (b) of removingsaid leachate from said leached feedstock comprises crushing saidleached feedstock to produce a pressate and a solid feedstock, saidpressate comprising soluble protein which is further processed foranimal feed.
 38. A method of producing ethanol comprising, in thisorder: a) preparing said composition comprising xylose and treatedfeedstock according to claim 1; b) treating said composition comprisingxylose and treated feedstock with cellulase under conditions whichhydrolyse cellulose in said treated feedstock to glucose, producing asugar solution comprising xylose and glucose; and c) fermenting saidsugar solution to ethanol.
 39. A method of producing ethanol,comprising: a) preparing said composition comprising xylose and treatedfeedstock according to claim 1; and b) treating said composition with amicroorganism under conditions which permit fermentation of said xylosein said composition to ethanol.
 40. A method of producing ethanolcomprising, in this order: a) preparing said composition comprisingxylose and treated feedstock according to claim 1; b) separating saidtreated feedstock from said composition; c) treating said treatedfeedstock with cellulase under conditions that hydrolyze cellulose insaid treated feedstock to glucose, producing a sugar solution comprisingglucose; and d) fermenting said sugar solution to ethanol.
 41. Themethod of claim 1, wherein leaching is performed at a temperature ofabout 60° C. to about 80° C.
 42. The method of claim 41, wherein said atleast one aqueous solution of step (a) comprises about 0.25 to about 10times the maximum water holding capacity per kilogram of drylingocellulosic feedstock.
 43. The method of claim 1, wherein leachingis performed for a period between about 10 minutes and about 30 minutes.44. The method of claim 42, wherein leaching is performed at atemperature of about 70° C.
 45. The method of claim 42, wherein theaqueous solution has a pH of about 6 to about
 7. 46. The method of claim42, wherein said leached feedstock is treated in step (c) at atemperature of about 100° C. to about 220° C.
 47. The method of claim46, wherein said leached feedstock is treated for about 5 seconds toabout 60 minutes.
 48. The method of claim 1, wherein said treatment ofstep (c) comprises a first stage at a temperature between about 100° C.to about 180° C. followed by a second stage at a temperature betweenabout 180° C. to about 270° C.
 49. The method of claim 48, wherein saidfirst stage is performed between about 0.25 to about 24 hours.
 50. Themethod of claim 49, wherein said second stage is performed between about5 to about 120 seconds.
 51. A method of producing xylose and glucosecomprising, in this order: a) leaching a mechanically-disrupted orpreconditioned lignocellulosic feedstock comprising greater than about20% (w/w) cellulose, said leaching being conducted prior to any chemicaltreatment of said lignocellulosic feedstock, comprising contacting saidfeedstock with at least one aqueous solution of pH 6 to 9 for a periodgreater than 2 minutes at a temperature of 25° C. to about 80° C. toproduce a leached feedstock and a leachate; b) removing the leachatefrom said leached feedstock; and c) treating said leached feedstock withan aqueous solution at a temperature of at least about 100° C. withammonia under conditions which disrupt fiber structure of the leachedfeedstock and hydrolyze a portion of hemicellulose in said leachedfeedstock to produce a composition comprising a treated feedstock; andd) treating said composition with an enzyme comprising cellulase underconditions which hydrolyze cellulose in said leached feedstock toglucose, thereby producing a sugar solution comprising xylose andglucose.
 52. The method of claim 1, wherein during step (b), betweenabout 20 % and about 100% of the leachate is removed from the leachedfeedstock.
 53. The method of claim 51, wherein said leaching isconducted for a period of between about 10 minutes and about 5 hours.